Abstract A survey was made of the concentrations of Ag, od, Cr, Cu, Mn, and Zn found in the sand crab, Emerita analoga, and the sand it inhabits from various points in the Monterey Bay area of California. Geographical differences in concen¬ trations were documented and correlations between these con¬ centration patterns and possible point sources of contaminant input associated with domestic, industrial, and agricultural effluents were drawn. In addition, the effect of local current patterns in the concentration of various heavy metal pollutants was suggested by the research. The results suggest that Emerita may be a useful bio¬ geographical marker of trace metal levels in the sandy beach environment. 1/0 Introduction Much work has been done since the early nineteenth century to establish trace metal concentrations in a variety of biological systems, including marine organisms. For example, J. F. John in 1814 demonstrated the presence of manganese in the carapace of the marine crustacean Potamobius fluviatilis. Within the last ten years, with the advent of atomic absorption spectroscopy, much more rapid and relatively more accurate investigation has been made possible. Most of the work to date has been concerned with the establishment of normal trace metal concentration levels within certain species and is cited in Goldberg's 1967 review of the literature. The goal of the work reported herein has been first to catalog the concentrations of certain trace metals normally occurring within the marine anamuran crustacean, Emerita analoga, and second to attempt to analyze for differences in metal concentrations possibly attributable to known point sources of contamination. Therefore, a biogeographical survey was accomplished with special note made of local contamination inputs. The Monterey Bay area was chosen for study, since it has a large number of possible trace metal inputs: 6 direct sewage outfalls (of primary or secondary treatment only); 6 rivers and sloughs, 3 of which traverse large agricultural areas, and at least 2 of which are used for sewage disposal; 3 major fishing and boating centers; and 2 relatively large urban areas, Santa Cruz and Monterey. 119 The metals chosen for study were silver, cadmium, zinc, copper, manganese, and chromium. According to Bowen (1966), all have either high or very high potential for pollution. These metals are currently being introduced into the environment at a rate exceeding their annual cycling out of the marine environment. Furthermore, all, with the ex¬ ception of copper, are highly toxic at relatively low con¬ centrations. Thus a geographic survey of the concentrations of these metals in a given species would seem to be of value both in terms of assessing the possible biological impact of point sources of contamination, and also in providing base¬ line data for an area expected to receive even higher inputs of trace metals in the future. The sand crab, Emerita analoga (Arthropoda, amomura) was chosen primarily because of its ubiquity in the sandy beaches which make up a major portion of the Monterey Bay coastline. Furthermore, these animals do not move great distances laterally (MacGinitie, 1938) and are thus potenti¬ ally good biological markers of local conditions. The study was limited to females only as they are very much larger, easier to collect, and may be individually analyzed for trace metal content. 2C Methods and Materials Collection The collection of Emerita from Monterey Bay was ac¬ complished during a five day period in late April 1971. The aim was to collect all of the organisms over as short a time span as possible to eliminate climatic and seasonal effects. Twenty three stations from Carmel to Santa Cruz were sampled (Fig. 1). Approximately 20 female Emerita analoga were taken from each beach station. While in the field, the eggs were separated from the ovigerous females and stored in separate acid-cleaned containers. Sediment samples from the surface of the sand were also taken from each of the twenty three stations. The samples of whole animals, eggs, and sediments were then frozen and stored until further use. In late May another set of Emerita were collected from Pt. Sur, and area believed to be relatively unpolluted, for comparison with the established base line of metal concentrations in the Emerita populations from around the Monterey Bay area. Whole Animal Procedure For each analysis, 5 to 8 animals per station were thawed, blotted dry, and a fresh, wet weight was recorded. The specimens were then dried at 80°C. for approximately 24 hours. A dry weight was recorded and each of the animals was then crushed into a fine powder, using a mortar and pestle (Thiers, 1957). Approximately 1/2 gram of this crushed material was digested using a modification of the Middleton¬ ( Stuckey wet digestion method (Christian and Feldman, 1970) suggested by Martin (personal communication). In this pro¬ cedure, 5 to 7 ml of 90% nitric acid is added to the crushed material. This suspension is refluxed for approximately two hours, allowed to evaporate to 2 ml, and then cooled. Approxi¬ mately 5 ml hydrogen peroxide is then added and the suspension again heated, refluxed, boiled down and diluted to 25 ml with distilled water. This was a most successful method for the removal of the residual organic material in Emerita. Charring was deemed both unnecessary and undesirable in light of pos¬ sible loss of volatile elements. There were noticeable amounts of sand-like particles remaining, but these were assumed to be unavoidable, recognizing the habitat and feeding patterns of Emerita, and were merely allowed to settle out of suspen¬ sion. A blank solution was run under the same conditions to account for any contamination of reagents and vessels. The solutions were then analyzed by atomic absorption spectro¬ scopy for the trace metals listed above, using a Perkin-Elmer model 303 Spectrophotometer (Kahn, 1968). The effect of light scatter on the analyses of Cd and Zn was corrected for by reading the absorption of each of the samples (2203 A° and adding this value to the absorption recorded for the blank run for each particular set. Data were calculated on the basis of ug/gram, or parts per million, dry weight (Christian and Feldman, 1970). ad Eggs Eggs were collected from the majority of the stations sampled. The eggs collected were all under Stage 5 of de¬ velopment (Eickstadt, 1969). These were dried for 24 hours at 80°C., and then weighed. The dried eggs were then sub¬ jected to basically the same wet digestion and atomic absorp¬ tion techniques outlined above for the whole animals with one exception. Eggs had a higher proportional amount of lipid per dry weight than whole animals. Consequently, several extra steps of refluxing with both nitric acid and hydrogen peroxide were needed. A blank was run concurrently to correct for contamination by reagents and vessels. Only one analysis could be made per station as a result of the very small dry weight of eggs available. Thus there was no possibility for statistical validation of the results obtained. Sediments Five sediment analyses were made per station; each sample an aliquot of a test tube of sand collected at the sand surface at each station. The wet digestion and atomic absorption techniques described above were followed throughout. The alternative of breaking down the sand granules themselves, using hydrofluoric acid was rejected on both practical and theoretical grounds. The crabs cannot be thought to be af¬ fected by the components of sand granules, nor can we expect that the trace metal concentrations in these components are substantially altered by trace metal contamination. Results and Discussion The results have been expressed as ppm dry weight. This was thought to be a more reliable figure than ppm wet weight, as the variance of the water content per sample was considerable. Based upon weight ratios, the ppm dry may generally be converted to ppm wet weight using the wet to dry ratio of 3.85 for animals and 1.26 for sediment. Wet weight figures for the eggs were not obtained. For each metal, the mean value in ppm for whole ani¬ mals and for sediments from each individual station was com¬ pared with that of every other station using the "student t" test. On the basis of this data, signficantly differing populations, based on statistically different concentration levels, were recognized and cataloged. These differences are displayed in Figure 2, a-f, through the use of different hashing marks on the maps of the coastline of the Bay. Note also that the mean values and standard deviations are re¬ corded to the left of each diagram. The results obtained for concentrations found along the coast will be discussed according to metal. Silver (Fig. 2a) has been documented as occuring in marine organisms at levels ranging from 3 to 11 ppm (Vinogradov, 1953), but Bowen (1966) lists no silver values as having been recorded for crustacea. The data reported herein lies in the range reported by Vinogradov showing a range of 1.4 to 4.2 ppm. According to Christian and Feldman (1970), silver is an environmental contaminant and a cumulative poison, inhibiting 24 enzymes in two ways. First, it competes with copper for position in certain metalloenzymes, and second, it reacts with thiol groups near enzymatic active sites, as does mercury. Harvey (1968) asserts that the major input of silver into biological systems is through adsorption, stating that silver is not present as a salt in seawater. Thus adsorption and food chain amplification may occur, and Emerita, found low on the food chain (eating mostly plankton -- Efford, 1967) may thus be expected to have relatively low values for this metal. Referring to Figure 2a, we see the highest silver content occurs at the southwestern tip of the Monterey Peninsula. Peters (1971) in her study of metal concentra¬ tions in Pagurus samuelis also noted the highest silver con¬ centrations in this anomuran to be in populations from the Monterey Peninsula, specifically from the area adjacent to the Pacific Grove sewer outfall. According to Vieth (1971) however, silver concentrations in the sewage effluent of Pacific Grove are relatively low as compared with those values found in the Monterey sewage effluent (under 5 ppm as compared to 25 ppm). The high values represented at this area in Emerita may be illustrative of current patterns which could bring plankton from a relatively more contaminated area for feeding or may merely represent some as yet unknown source of input. Note however that the second most highly concentrated area for silver was from the area near the Monterey sewer effluent. The high levels seen in animals from the northern tip of Monterey Bay near Santa Cruz remains unexplained. 125 Cadmium has been found in marine organisms at levels from .15 to 3 ppm (Mullin and Riley, 1956). Again, no crus¬ tacean data is presented by Bowen (1970) for this element, but Brooks and Rumsby (1965) report finding cadmium in the proteins of certain molluscs. It has been found to be a cumulative poison (Vinogradov, 1953) and Christian and Feldman (1970) report that cadmium does inhibit certain enzymes. It has been recorded occurring least in calcarious tissue of exo¬ skeleton, perhaps indicating that adsorption is not the major factor in cadmium uptake. Figure 2b shows that the cadmium concentrations found in Emerita fall in the range already reported for marine organisms. Cadmium values were the highest in the central Monterey Bay area, perhaps indicative of con¬ tamination from the Moss Landing or Salinas River area. Tolan (1971) documented possible biological contamination here in his finding of increased cadmium values in molluscs and crustacean found in the Moss Landing area. It is interest¬ ing to note the higher values occur in the central area of the Bay, an area more highly influenced by river and agri¬ cultural inputs and removed from the urban industrial and domestic sewage outfalls. Chromium is an essential element at trace levels. It has been found to activate the enzyme phosphoglucomutase (Strickland, 1949). However it is highly toxic at higher levels. Bowen reports that Cr-IV is very much more toxic than Cr-III, perhaps even carcinogenic. Chromium has been documented in marine animals and occurs at concentrations of 126 from .2 to 1 ppm (Bowen, 1970); Fukai and Fukai and Broquet (Goldberg, 1967) documented chromium concentrations in Eriphia verrucosa and Pisa nodipus, two crustacean of 1.1 and .48 ppm respectively. In Figure 2c it can be seen that in only one location did my data correspond within one order of magnitude to these figures. Error due to contamination of vessels and reagents was at a minimum and corrections were made by means of a blank, therefore, confidence may be placed in this data. The high figures of 40 to 50 ppm found in the Monterey Basin may be illustrative of pollution due to chro¬ mium boat paint used in the harbor, domestic sewage, or in¬ dustrial input into the Bay which congregates in the basin to the southeast around the city of Monterey. The relatively high levels found around the Asilomar-Pt. Pinos area may re¬ inforce the earlier contention that local current systems may move pollutant from the basin around the point. In light of the fact that Emerita from the southern tip of the Peninsula were uncontaminated, these higher values probably represent contamination from the Pacific Grove sewage effluent. Copper is found in all animals. It is a component of metalloenzymes: many having an oxidative role. It is also a component of heamocyanin, the crustacean blood pigment. It is, however, according to Bowen (1970), highly toxic at relatively low levels. Major pollution sources are mining operations, copper piping and fungicides in soil runoffs. Levels reported by Vinogradov (1953) in marine animals ranger / from 4-50 ppm; Culkino and Riley (Goldberg, 1967) cite a value of 35.0 ppm in whole animal Cancer pagurus, another decapod. The Emerita base line data (Fig. 2d) for copper, reported here, is not higher than recorded elsewhere. It is interesting to note the extremely high values recorded at the Seaside and Fort Ord sewers which correlate directly with Vieth's findings of high copper concentrations in the actual effluents of the Seaside and Monterey sewage plants. Noting the high values in the northern pocket of the Bay, one is tempted to associate these increased copper values with urban marine contamination taking place from the major population centers at the north and south ends of the Bay. Again we see relatively high values on the Monterey Peninsula; perhaps indicative of either local current struc¬ ture or sewage contamination from Pacific Grove. Man ganese is an essential element to physiological functions. It has been found to be an enzyme activator for many of the enzymes in the Krebs Cycle. It is also a con¬ stituent of many metalloenzymes found in the blood. It competes with magnesium for position in certain other metal¬ loenzymes and thus, in higher concentrations, is an enzyme inhibitor. Levels reported in the literature for marine animals range from 1 to 60 ppm. The data presented in Figure 2 fall in this same range. Noting the lower solubility of manganese in salt water over fresh water, it is interesting to see that the highest Mn values found in Emerita were found at each of the river inputs to the Bay. Manganese has been 28 reported to be found in very high concentrations in calcareous tissues. In view of this, and noting the wide standard devia¬ tions exhibited, one may suspect that the molt state of the animal may be a factor in the manganese concentrations. No correlations with molt state of Emerita were performed. Zinc is an element found in all organisms. It is a component of many metalloenzymes and proteins, toxic at higher levels in the environment. It has been found in marine or¬ ganisms at concentrations from 6 to 1500 ppm. Hiyama (Goldberg, 1967) found crustacea to have a concentration factor of well over 500, indicating the zinc concentration in crustacea should be over 5 ppm. The values reported herein ranged from 44 to 87 ppm. Not much deviation is seen in zinc concentra¬ tions. However, statistically differing concentration levels were easily recognized; the highest concentrations being found in Carmel Bay for which no explanation is drawn. Noting that metals may act in a synergistic fashion in either inhibition or activation of enzyme systems (Bowen, 1970), the relative concentration levels of metals were plotted against each other for the area (Fig. 3). Actual ppm figures are not comparable between the metals, but note the relative levels found in the animals at the various lo¬ cations. The heights of the bars and the distinct hashing types refer to the statistically differing ppm levels ex¬ pressed in Figure 2 for each metal. Also in this diagram are recorded the levels found in Emerita from Pt. Sur. for silver, manganese, chromium and zinc, the concentrations corresponded with the concentrations in populations having relatively lower amounts of these metals. However for copper, and more strikingly, in the case of cad¬ mium the values found at Pt. Sur are relatively high. The extent to which this may be illustrative of natural variability, contrary to contamination due to pollution factors, such as a nearby naval facility, is unknown. Figure 4 represents a geographical comparison of metals found in the sediments along the coastline. Silver and cadmium were not found in any of the sediments at a value above the detection limit of the spectrophotometer. No sta¬ tistical correlation could be found between metals. However, in scanning the data one may perceive a general correlation. All four metals seem to concentrate in the Monterey basin area (Stations 4 and 5). Also, the Salinas and Pajaro River areas (Stations 10 and 15) seem to be areas of concentrations. The hypothesis in this work has been that sand is very much more transient than the animals studied. The values recorded for metal concentrations in sediment may be illustrative then of current patterns and sand shifting. Metal concentration may be an effective means of cataloging sediment translocation. By plotting dry weight against ppm found per animal, no positive or negative correlation could be found with any of the metals. Several problems exist in expressing the egg data: eggs were not found at all of the stations, there was much 30 lipid still undigested after several refluxing steps, there was no chance for statistical validation of differences, and finally, the dry weight for the aliquot used was so small that my ppm calculations would be subject to a great degree of error. However, mean values for each of the Bay area egg samples are listed below: Standard deviation Mean Cd no detectable amounts .89 .38 Ng 8.82 37.73 Zn 5.9 8.5 Mn 2.63 5.1 18.39 Cr 8.87 13 Acknowledgements I would like to gratefully acknowledge the invaluable assistance and advice given me by Dr. John Martin and Dr. Welton Lee. Thanks is extended to Dr. John Phillips for his help in statistical analysis. Literature Cited Bowen, H. J. M. (1966). Trace Elements in Biochemistry. Academic Press, London. 241 pp. Brooks, R. R. and Rumsby, M. G. (1965). Limnol. Oceanogr. 10: 521-528. Christian, G. D., and Feldman, F. J. (1970). Atomic Absorp¬ tion Spectroscopy. Applications in Agriculture, Biology and Medicine. John Wiley and Sons, N.Y. 490 pp. Efford, I. (1967). Feeding in the sand crab Emerita analoga. Crustaceana 10(2): 167-182. Eickstadt, L. L. (1969). The reproductive biology of the sand crab Emerita analoga. Doctoral dissertation, Hopkins Marine Station of Stanford University. Goldberg, E. D. (1967). Review of Trace Element Concentra¬ tions in Marine Organisms. Puerto Rico Nuclear Center. 535 pp. Harvey, H. W. (1968). Biological Oceanography. in Recent Marine Sediments, a symposium. P.D. Trask, ed. Dover Publications. N.Y. 736pp. Kahn, Herbert (1968). Principles and Practice of Atomic Absorption Spectroscopy. in Trace Inorganics in Water, A symposium sponsored by the Amer. Chem. Soc., Washington, D.C. 396 pp. MacGinitie, G. E. (1938). Movements and Mating habits of the sand crabs, Emerita analoga. Amer. Midl. Natl. 19: 471-481. /3. Mullin, J. B. and Riley, J. P. (1956). The occurrence of cadmium in seawater and in marine organisms and sediments. J. Mar. Res. 15: 103-115. Peters, J. (1971). Trace Metals in the marine intertidal hermit crab, Pagurus samuelis in California waters. On file Hopkins Marine Station, Pacific Grove. Strickland, L. H. (1949). The activation of phosphogluco¬ mutase by metal ions. Biochem. J. 44: 190-198. Thiers, R. E. (1957). Contamination in trace element analysis and its control. in Methods of Biochemical Analysis. V.5. D. Glick, ed., Interscience, N.Y. 502 pp. Tolan, T. (1971). Trace metals in common marine foods. On file at Hopkins Marine Station, Pacific Grove. Vieth, R. (1971). Trace metals in Monterey Peninsula sewerage. On file at Hopkins Marine Station, Pacific Grove. Vinogradov, A. P. (1953). The Elemental Chemical Composition of Marine Organisms. Sears Foundation for Marine Research, New Haven. 535 pp. 3 C O C FIGURE 1. of the Monterey Bay Area showing collection sites. 13. 23 Santa Cruz + 20 2221 8 3 24 Monterey Carmel CarmelR. 17 136 Watsonville 16 15 Moss Land 12 17 10 SalinasRive 8 Sewer Outfalls 6 Monterey 7 Seaside 8 FtOrd 9 Marina C FIGURE 2a. Silver concentrations found in Emerita from the Monterey Bay area. Mean values and standard deviations for each station are shown at left. Significantly differing populations are indicated by distinct hash marks noted along the coastline. yearly aupploments and weekly Notices to not to tow submerged objects across the transit lane Mariners. Copies of the regulations may when it is in use. +045 be obtained at the office of the District Engineer, Corps of Engineers in San Fran¬ Soquel cisco, Calif. P OTAN Anchorage Regulations may be obtained Cappo at the office of the Commander, 12th Coast g Guard District in San Francisco, Calif. ereg Refer to section numbers shown with 0 area designation. Watsonville . PRedsandHil PR. TRS. VOON 1460 k42) SILVER -2.5 ppm 57 hor 2.5 -2.75 o wo oss Landing Karber Lag. O 2.76-4.0 nvnille 4. - 5 aro sohnes OIR. TR r EE S Rot W&c . a 20 S MONTER Veande Monte Monterey Peninsula Rot WaG A . 22 oma a Va. Carmel 138 1 C FIGURE 2b. Cadmium concentrations in Emerita from the Monterey Bay area. Mean values and standard deviations for each station are shown at left. Significantly differing populations are indicated by distinct hash marks noted along the coastline. C vearly supplements and weekly Notices not to tow submerged objects across the transit lane Mariners. Copies of the regulations may when it is in use. +045 be obtained at the office of the District Engineer, Corps of Engineers in San Fran¬ Squl cisco, Calif. aor Anchorage Regulations may be obtained T OTANK at the office of the Commander, 12th Coast pg Son es Guard District in San Francisco. Calif. 1 Refer to section numbers shown with area designation. eonalle RRedsandHil R. TRS. KOON 146662). CADMIUM -2.5ppm KS(S OF TWO) 2.51 - 3.0 Moss Landing Herber o.. Lag. O . 3.0 -3.52 ano ones (IDD 630 KH2) AERO Rot W & G dei Se A MONTENE enode Del Monte Monterey Peninsula Rera Loma in larmel S TOWERCMELTE OT 040 140 0 FIGURE 2c. Chromium concentration found in Emerita from the Monterey Bay area. Mean values and standard deviations for each station are shown at left. Significantly differing populations are indicated by distinct hashmarks noted along the coastline. yearly supplements and weekly Notie not to tow submerged objocts across the transit lane Mariners. .Copies of the regulations may when it is in use +015 be obtained at the office of the District Engineer, Corps of Engineers in San Fran¬ Soquel cisco. Calif. OTANK Anchorage Regulations may be obtained Capog at the office of the Commander, 12th Coast ranay L cnr. Guard District in San Francisco, Calif. CRUZ PLakes Ver Reter to section numbers shown with n area designation. Watsonville RrdsandHl a CHROMIUM 0- ppm 2 S 3-30 STACKS(S OF TWO oss Landing Karber Mo. Lag. OTRR 7 31-40 tville 41-53 o e O r. (KIDD63O KH2) Marina AERC Rot wamc n0 120 o Kn. Del Monte Montorey Peninsula Rot W & G . 22 Laa S Carmel . oregeneter vongren, 040 5TOHE N0. e O FIGURE 2d. Copper concentrations found in Emerita from the Monterey Bay area. Mean values and standard deviations for each station are shown at left. Significantly differing populations are indicated by distinct hash marks noted along the coastline. yearly supplements and wookly Notices to not to tow submerged objects across the transit lane Mariners. Copies of the regulations may when it is in use +045 be obtained at the office of the District Engineer, Corps of Engineers in San Fran¬ Squel cisco, Calif. g Anchorage Regulations may be obtained OTANI Coaa at the office of the Commander, 12th Coast Aptor ara Guard District in San Francisco, Calif. en Reier to section numbers shown with area designation. snvile ORed Sand Hill R. TRS AKPON 14602) COPPER 40-60 ppm o Eehons ACKS(S O 61-70 oss Landing Harber Moss Lag. OTAR 71-90 e 91-110 ooen O/R. TR (KIDD630 KH2) PNarn. AERO Rot W& G re endde Del Monte Monterey Peninsula 230 Rot Wap;G 2 . 2 loma ONEEICANNEUTE NONASTERN 1 600 STONE HO. 6 j O FIGURE 2e. Manganese concentrations in Emerita found in the Monterey Bay area. Mean values and standard deviations for each station are shown at left. Significantly differing populations are indicated by distinct hash marks noted along the coastline. or vearly supplements and woekly Notices to not to tow submerged objects across the transit lane Mariners. Copies of the regulations may when it is in use be obtained at the office of the District 1015 Engineer, Corps of Engineers in San Fran¬ Squel + cisco, Calif. Anchorage Regulations may be obtained OTAN C at the office of the Commander, 12th Coast Pgs. Ptan Guard District in San Francisco, Calif. Grdat eeg Refer to section numbers shown with area designation. d . Watsonville RedSand (OON 1460R2) MANGANESE I1-20ppm 21 45 DSTACKS(S OF TWO 2 Moss Landing Harber Moss Lag. OTANK 71 46 i- Saso ohes Je. r. KIDD63O KHD Marina AERC Rot W & ( essde Monterey Peninsula Del Monte Rot W & G SIACK ona h. Dan. i care TONERCANELITE NONASTERN) 200 STONE NO. 146 C FIGURE 2f. Zinc concentrations found in Emerita from the Monterey Bay area. Mean values and standard deviations for each station are shown at left. Significantly differing populations are indicated by distinct hash marks noted along the coastline. yearly suppléments and weekly Notices to not to tow submerged objects across the transit lane Mariners. Copies of the regulations may when it is in use. +015 be obtained at the office of the District Engineer, Corps of Engineers in San Fran¬ Squel ) cisco. Calif. ajors TANK Anchorage Regulations may be obtained C a A at the office of the Commander, 12th Coast . Guard District in San Francisco, Calif. Refer to section numbers shown with area designation. . Watsonville Red Sand Hill R. 7RS. KOON 1460 KH2) TINC 40-50 ppm 2. 51-70 STACKS(S OF TWO Moss Landing Harber o 14 OTAN . 71-80 89 81 SANO GUNALR Oe. 72 630 K2) AM ER Rot W&C 240 k MONTERE . Dyl Monte Monterey Peninsula Rot W & G n Loma ai Carmel QOVERICMRNELITE MONASTERN) o0 116 C C FIGURE 3. Relative concentrations of metals found in Emerita, Stationn ber. Distinct hashing organi types refer to significantly differing populations. O = ce 150 0 FIGURE 4. Relative concentrations of metals found in sediments, organized by Station number, with corresponding absolute values listed beneath. O (OO O 00 O NO C — C C O O O O 3 0 O N 5